PROJECT SUMMARY Integration is essential for HIV-1 replication and is completed by integrase (IN). A class of drugs which inhibit the strand transfer (ST) function of HIV integrase, called IN strand transfer inhibitors (INSTIs), includes approved drugs raltegravir (RAL), elvitegravir (EVG) (1st generation) and dolutegravir (DTG), bictegravir (BIC) (2nd generation). DTG has a higher genetic barrier to resistance than RAL or EVG, and is recommended by the World Health Organization as an alternative to efavirenz in first-line regimens in low- and middle-income countries (LMICs). Selection for DTG resistance is rare, but does exist and is currently not well understood. There is mounting evidence for failure of DTG-based treatment in clinical trials (VIKING-3 study) in the absence of mutations in the targeted IN gene. Our overarching hypothesis is that mutations outside IN can impart drug resistance to IN-targeting drugs through indirect interactions that we call epistatic. The scientific premise for studying these interactions is soundly grounded on two key pieces of evidence: First, in surprising preliminary data from in vitro serial passage experiments in the presence of increasing amounts of DTG, a DTG resistance mutation located outside IN was discovered. Experiments with recombinant viruses validated DTG resistance of this mutation and showed enhanced resistance in the presence of the E157Q IN polymorphism. Moreover, deep sequencing analyses showed that compared to infection by wild-type, mutant?containing viruses resulted in more insertions, deletions, and non-canonical long terminal repeat (LTR) ends in 2-LTR circles and integrated viral DNA. Second, a recent independent study based on similar serial passage experiments, identified changes at the general G-tract region of the 3?-polypurine tract (3?-PPT) in a DTG-resistant virus (Malet et al., 2017). Subsequently, different 3?-PPT changes were reported in a patient that failed DTG therapy. However, the mechanism of DTG resistance through mutations at the 3?-PPT remains unclear due to conflicting hypotheses and lack of experimental validation. Our hypothesis is that mutations outside IN can affect DTG resistance by altering the LTR ends at the INSTI binding site. This hypothesis will be tested by a team of experts that includes PIs Sarafianos (biochemical, virological drug resistance mechanisms), PI Lyumkis (single particle cryo-EM on intasome/drug complexes) and PI Hachiya (virology, drug resistance) with the support by HIV IN experts Hughes (NCI) and Kvaratskhelia (U Colorado), using virological, biochemical, and structural tools to address the aims to investigate the virological, biological, and structural mechanisms of DTG resistance through epistatic interactions. These innovative studies will help elucidate the molecular mechanisms of INSTI resistance through epistatic interactions via mutations that are outside the IN gene and affect the INSTI-binding site. They are significant and will help explain clinical failures to DTG-based regimens in the absence of mutations in IN.